A universal combinatorial design of antibody framework to graft distinct CDR sequences: a bioinformatics approach.

Antibody (Ab) humanization is crucial to generate clinically relevant biologics from hybridoma-derived monoclonal antibodies (mAbs). In this study, we integrated antibody structural information from the Protein Data Bank with known back-to-mouse mutational data to build a universal consensus of framework positions (10 heavy and 7 light) critical for the preservation of the functional conformation of the Complimentarity Determining Region of antibodies. On the basis of FR consensus, we describe here a universal combinatorial library suitable for humanizing exogenous antibodies by CDR-grafting. The six CDRs of the murine anti-human EGFR Fab M225 were grafted onto a distinct (low FR sequence similarity to M225) human FR sequence that incorporates at the 17 FR consensus positions the permutations of the naturally observed amino acid diversities. Ten clones were selected from the combinatorial library expressing phage-displayed humanized M225 Fabs. Surprisingly, 2 of the 10 clones were found to bind EGFR with stronger affinity than M225. Cell-based assays demonstrated that the 10 selected clones retained epitope specificity by blocking EGFR phosphorylation and thus hindering cellular proliferation. Our results suggest that there is a universal and structurally rigid near-CDR set of FR positions that cooperatively support the binding conformation of CDRs.

Generation and characterization of human anti-human IL-21 neutralizing monoclonal antibodies.

Interleukin-21 (IL-21) is a type I four-helical bundle cytokine that exerts a variety of significant effects on many hematopoietic cells, including T and B lymphocytes and natural killer cells. IL-21 is produced predominantly by CD4+ T cells and natural killer T cells and, when aberrantly overexpressed, appears to play important roles in a wide variety of autoimmune disorders. To generate potential therapeutic reagents capable of inhibiting IL-21 for clinical use, we immunized human immunoglobulin transgenic mice with IL-21 and then identified and cloned a panel of human anti-human IL-21 binding monoclonal antibodies. IL-21 neutralizing and IL-21-binding, non-neutralizing antibodies were assigned to distinct epitope "bins" based on surface plasmon resonance competition studies. The most potent neutralizing antibodies had extremely high (sub pM) affinity for IL-21 and were able to block IL-21 activity in various biological assays using either an IL-21R-transfected pre-B-cell line or primary human B cells, and their neutralizing activity was, in some cases, superior to that of a soluble form of the high affinity heterodimeric IL-21 receptor. Characterization of this panel of IL-21 antibodies provided the basis for the selection of a therapeutic candidate antibody capable of inhibiting IL-21 activity for the treatment of autoimmune and inflammatory diseases.

Therapeutic bispecific antibodies: The selection of stable single-chain fragments to overcome engineering obstacles.

The clinical success of mAbs continues to reinforce antibody engineering as an essential tool for the development of biologics. Research focused on discovering the next generation of therapeutics has prompted a revisiting of the concept of bispecific antibodies (bsAbs). Recently, clinical programs investigating combinations of mAb therapies have renewed interest in the applications of bsAbs. However, because of challenges with production, efforts directed toward the development of bsAbs have yet to yield a product approved by the FDA. The current status of these proteins implies that the strategies for constructing therapeutic bsAbs will likely require a highly refined design plan at the outset of the engineering process. Antibody fragments are attractive building blocks for the assembly of bsAbs. Of the recombinant antibody fragments, single-chain variable fragments (scFvs) offer the advantage of expression as a single polypeptide, thereby greatly simplifying production. However, issues with stability have plagued these proteins and limit the application of scFvs as therapeutics. Recent advances in selection processes using display platforms have been reported that facilitate the 'evolution' of scFvs to obtain stabilities comparable with those of mAbs. The timely advances in scFv engineering parallel the resurgence of bsAbs and enable the construction of dual-targeting proteins that can be manufactured as therapeutics.

A dual-targeting PDGFRbeta/VEGF-A molecule assembled from stable antibody fragments demonstrates anti-angiogenic activity in vitro and in vivo.

Targeting angiogenesis is a promising approach to the treatment of solid tumors and age-related macular degeneration (AMD). Inhibition of vascularization has been validated by the successful marketing of monoclonal antibodies (mAbs) that target specific growth factors or their receptors, but there is considerable room for improvement in existing therapies. Combination of mAbs targeting both the VEGF and PDGF pathways has the potential to increase the efficacy of anti-angiogenic therapy without the accompanying toxicities of tyrosine kinase inhibitors and the inability to combine efficiently with traditional chemotherapeutics. However, development costs and regulatory issues have limited the use of combinatorial approaches for the generation of more efficacious treatments. The concept of mediating disease pathology by targeting two antigens with one therapeutic was proposed over two decades ago. While mAbs are particularly suitable candidates for a dual-targeting approach, engineering bispecificity into one molecule can be difficult due to issues with expression and stability, which play a significant role in manufacturability. Here, we address these issues upstream in the process of developing a bispecific antibody (bsAb). Single-chain antibody fragments (scFvs) targeting PDGFRbeta and VEGF-A were selected for superior stability. The scFvs were fused to both termini of human Fc to generate a bispecific, tetravalent molecule. The resulting molecule displays potent activity, binds both targets simultaneously, and is stable in serum. The assembly of a bsAb using stable monomeric units allowed development of an anti-PDGFRB/VEGF-A antibody capable of attenuating angiogenesis through two distinct pathways and represents an efficient method for rapid engineering of dual-targeting molecules.

Engineering of stable bispecific antibodies targeting IL-17A and IL-23.

Bispecific antibodies (bsAbs) present an attractive opportunity to combine the additive and potentially synergistic effects exhibited by combinations of monoclonal antibodies (mAbs). Current challenges for engineering bsAbs include retention of the binding affinity of the parent mAb or antibody fragment, the ability to bind both targets simultaneously, and matching valency with biology. Other factors to consider include structural stability and expression of the recombinant molecule, both of which may have significant impact on its development as a therapeutic. Here, we incorporate selection of stable, potent single-chain variable fragments (scFvs) early in the engineering process to assemble bsAbs for therapeutic applications targeting the cytokines IL-17A/A and IL-23. Stable scFvs directed against human cytokines IL-23p19 and IL-17A/A were isolated from a human Fab phage display library via batch conversion of panning output from Fabs to scFvs. This strategy integrated a step for shuffling V regions during the conversion and permitted the rescue of scFv molecules in both the V(H)V(L) and the V(L)V(H) orientations. Stable scFvs were identified and assembled into several bispecific formats as fusions to the Fc domain of human IgG1. The engineered bsAbs are potent neutralizers of the biological activity of both cytokines (IC(50) < 1 nM), demonstrate the ability to bind both target ligands simultaneously and display stability and productivity advantageous for successful manufacture of a therapeutic molecule. Pharmacokinetic analysis of the bsAbs in mice revealed serum half-lives similar to human mAbs. Assembly of bispecific molecules using stable antibody fragments offers an alternative to reformatting mAbs and minimizes subsequent structure-related and manufacturing concerns.